PROJECT SUMMARY Cell culture technology is at the heart of all fundamental cell biology research. Disease modeling, stem-cell research, drug development, and tissue engineering all rely heavily on the in vitro culture of cells. Cells isolated or grown in the laboratory are oftentimes maintained in cell culture vessels made of glass or plastic. These vessels lack the structural, mechanical, and chemical microenvironmental cues that cells use to regulate processes as diverse as cell signaling, differentiation, division, and even life and death. These cues originate from the extracellular matrix (ECM), which exhibits a range of essential chemical and mechanical properties that vary widely from organ to organ. The failure of traditional cultureware to reproduce these vital ECM cues is a significant contributor to the non-physiologic structural, phenotypic, and functional development of in vitro cell cultures, impairing the predictive in vitro assessment of cellular responses to drugs and pathological challenge. Researchers employ a variety of approaches to enhance the physiologic relevance of in vitro cell culture environments toward platforms that better resemble native cellular niches. Many of these techniques are a challenge to implement; they require expensive fabrication equipment and manual labor and demand a trade-off between scalability and biological complexity. Further, these approaches can suffer from significant failure rates and poor reproducibility. Customizing the structural and mechanical properties of cell culture niches is time- consuming, making it a challenge to adapt these culture niches to new model systems. NanoSurface Biomedical proposes to develop a platform based on roll-to-roll nano-imprint lithography (R2RNIL) capable of fabricating customizable culture substrates with biomimetic nanoscale topographies at scale. While R2RNIL has been used extensively in the nanoelectronics industry, it has not yet been adapted to manufacturing processes for cell biology applications. Over the course of this project, the company will design and fabricate a custom production- ready R2RNIL machine with components tailored to the production of biocompatible substrates with biomimetic microenvironmental architectures. This novel R2RNIL platform will offer facile adjustment of the shape, orientation, and mechanical properties of the printed substrate material. Through these efforts, the company will produce a first-and-only platform for the high-throughput fabrication of nanoscale biomimetic cell culture niches that can be easily adapted to a variety of applications. Culture niches that recapitulate the shape and structure of the extracellular environment of heart and bone tissue will be fabricated. Cells grown on these substrates are expected to show phenotypes that are more representative of human physiology, increasing the predictive power of many cell-based assays. This work will serve as the basis for progression into Phase 2, where a wider-variety of cell culture niches and novel tunable substrates will be developed.